Live vaccines against gram-negative pathogens, expressing heterologous O-antigens

ABSTRACT

The present invention relates to live attenuated gram-negative vaccine carrier strains which are useful for expression and delivery of heterologous O-antigens (O-PS) from gram-negative pathogens. Said strains are deficient in the expression of homologous O-PS due to a defined genetic modification, preferably a deletion, and, thus, capable of efficiently expressing a desired heterologous O-PS in such a way that it is covalently coupled either to homologous or heterologous LPS core lipid A. The present invention furthermore relates to live vaccine carrier strains containing a heterologous gene or a set of heterologous genes encoding O-PS. Preferably, said strains additionally contain genes necessary for the synthesis of complete smooth heterologous LPS. The present invention also relates to live vaccines comprising said strains, preferably for immunization against gram-negative enteric pathogens.

This application is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No. PCT/EP96/04334, which has an Internationalfiling date of Oct. 4, 1996, which designated the United States ofAmerica, the entire contents of which are hereby incorporated byreference.

The present invention relates to live attenuated gram-negative vaccinecarrier strains which are useful for expression and delivery ofheterologous O-antigens (O-PS) from gram-negative pathogens. Saidstrains are deficient in the expression of homologous O-PS due to adefined genetic modification, preferably a deletion, and, thus, capableof efficiently expressing a desired heterologous O-PS in such a way thatit is covalently coupled either to homologous or heterologous LPS corelipid A. The present invention furthermore relates to live vaccinecarrier strains containing a heterologous gene or a set of heterologousgenes encoding O-PS. Preferably, said strains additionally contain genesnecessary for the synthesis of complete smooth heterologous LPS. Thepresent invention also relates to live vaccines comprising said strains,preferably for immunization against gram-negative enteric pathogens.

BACKGROUND OF THE INVENTION

Gram-negative enteric pathogens are the cause of a variety of diseasespresenting with a broad spectrum of symptoms ranging from mild waterydiarrhea to severe life-threatening symptoms such as fever, bloodydiarrhea, perforation or ulceration of the stomach or intestine, aloneor in combination. Examples of such diseases include typhoid fever,shigellosis, cholera, infections with enterotoxinogenic,enteropathogenic, and enterohemorragic Escherichia coli, and infectionswith Heliobacter pylori and Campylobacter jejuni.

The first stage of the infectious process occurs at the mucosal surfacewithin the digestive tract. Thus, interfering with this initial stage ofinfection prior to the onset of symptoms offers a particularlyattractive approach. The most effective means by which to accomplishthis would be to evoke a local protective immune response through theuse of an orally administered vaccine (Mestecky, J. Clin. Immunol. 7(1987), 265-276; McGhee and Kiyono, Infect. Agents Dis. 2 (1993), 55-73;Walker, Vaccine 12 (1994), 387-400). At present, 2 live oral attenuatedvaccines against enteric disease have been licensed for human use thesebeing the Ty21a strain of Salmonella typhi for the prevention of typhoidfever and the CVD103-HgR strain of Vibrio cholerae for the prevention ofcholera (Germanier and Furer, J.Infect.Dis, 131 (1975), 553-558; Levineet al., Lancet ii (1998), 467-470).

There exists a large body of evidence indicating that protection againstseveral enteric pathogens, such as S. typhi, E. coli, and Shigellaspecies is associated with the induction of an immune response againstcell surface components, specifically the O-antigen moiety of LPS,commonly referred to as O-polysaccharide (O-PS). For example, immunityto shigellosis, subsequent to recovery from either naturally-acquired orexperimentally-induced disease is correlated with a substantial rise inserum serotype-specific anti-LPS antibodies (DuPont et al.,J.Infect.Dis. 125 (1972), 5-11; DuPont et al., J.Infect.Dis. 12 (1972),12-16; Herrington et al., Vaccine 8 (1990), 353-357). Furthermore,epidemiological studies have also found that protection against Shigellainfections in the field was associated with increased levels of serumanti-LPS antibodies (Cohen et al. , J.Infect.Dis. 157 (1988),1068-1071). High levels of serum antibodies against Shigella LPS can bedetected among individuals residing in areas where such species ofShigella are endemic, presumably acquired by natural exposure and/orinfection with these pathogens.

LPS is an essential constituent of the gram-negative outer membrane andmay account for up to 70% of the cell surface components. LPS iscomposed of 3 regions: the innermost being lipid A which is embeddedinto the phospholipid outer membrane bilayer. The core polysaccharide isattached to the lipid A moiety usually via 2-keto,3-deoxyoctonate (KDO).The core is usually comprised of 5 to 7 sugars. To date, 7 types of coremolecules have been identified within the Enterobacteriaceae family andhave been named Ra, R1, R2, R3, R4, K-12, and B. Compared with theEnterobacteriaceae, V. cholerae possess an unusual core structure inthat it contains fructose and a single KDO molecule in the inner core(Kondo et al., Carbohydrate Res. 231 (1992), 55-64). The biosynthesis ofthe LPS core is encoded by the rfa locus. Among the Enterobacteriaceae,the rfa and rfb loci appear to be unlinked. In contrast, some evidenceexists to suggest a close linkage of at least part of these two loci forV. cholerae (Manning et al., p. 77-94. In Vibrio cholerae and Cholera:molecular to global perspectives (1994). Wachsmuth K., Blake, P. A., andOlsvik Y. (eds.). Washington, D.C.: American Society for Microbiology).

The outermost portion of the LPS molecule is composed of the O-PS whichconsists of repeating saccharide units of variable length (Luderitz etal., Curr. Top. Membr. Trans. 17 (1982), 79-151; Raetz, Annu. Rev.Biochem. 59 (1990) 129-170). The O-PS region of the LPS molecule confersserospecificity to the bacteria. The LPS molecule interacts closely withother molecules expressed on the outer membrane surface such as porinsand other outer membrane proteins (OMP), which determine thepermeability of the outer membrane. It is known that the assembly of OMPas well as secretion of proteins from the cell is affected by mutationsin the LPS of E. coli (Laird et al., J. Bacteriol. 176 (1994),2259-2204; Stanley et al., Mol. Microbiol. 10 (1993) 781-787).

Serospecificity is conferred not only by the sugars present in the O-PSbut also by their chemical linkage and sequence (Lüderitz et al., Curr.Top. Membr. Trans. 17 (1982), 79-151). Therefore, the O-PS is highlyvariable between gram-negative bacterial species whereas the corepolysaccharide is relatively constant within a given species or genera(Lüderitz et al., Curr. Topics in Membranes and Transport 17 (1982),79-151; Jansson et al., Eur.J.Biochem. 115 (1981), 571-577). Forexample, the genus Shigella includes a total of 47 known serotypesdivided among the 4 predominant pathogenic species which are S.dysenteriae (subgroup A, 12 serotypes), S. flexneri (subgroup B, 13serotypes) S. boydii (serogroup C, 18 serotypes) and S. sonnei (subgroupD; 1 serotype) (Ewing, In: Ewing W H, ed. Edwards and Ewing'sidentification of Enterobacteriaceae fourth edition. New York: ElsevierSci. Publish. Comp. (1986), 135-172). For example, in S. sonnei, theO-PS consists of a repeated disaccharide unit with 2 unusual sugars,2-amino-2-deoxy-L-alturonic acid linked to2-acetamido-4-amino-2,4,6-trideoxy-D-galactose by a 1,4 linkage (Kenneet al., Carbohydrate Res. 78 (1980), 119-126). In contrast, the O-PS ofserotype 1 of S. dysenteriae (which is the most common cause ofdysentery) is composed of repeating blocks ofrhamnose-rhamnose-galactose-N-acetylglucosamine (Ewing and Lindberg, In:Bergan T. (ed) Methods in microbiology vol.14. , Academic Press, London,pp. 113-142). The O-PS of V. cholerae 01 is comprised of 17-18perosamine subunits each of which is acylated with3-deoxy-L-glycero-tetronic acid. Quinovosamine has also been found inlow concentrations but its location within the O-PS of V. cholerae 01 isunknown (Redmond, FEBS Lett. 50 (1975), 147-149; Kenne et al.,Carbohydrate Res. 100 (1982), 341-349).

The enzymes involved in the biosynthesis of enterobacterial O-PS arecoded for by the rfb locus. In the case of Shigella species, anadditional gene, termed rfc, encodes the O-PS polymerase which functionsto polymerize the individual repeat units into chains of varying length.In most Shigella species, the rfb/rfc loci are located on the chromosome(Klena and Schnaitman, Microbiol. Rev. 57 (1993), 655-682). However, insome species of Shigella, all or part of the rfb locus is located on aplasmid episome (Maurelli and Sansonetti, Ann. Rev. Microbiol. 42(1988), 127-150). An additional gene, termed rfe, which is involved inthe synthesis of the enterobacterial common antigen (ECA) is alsorequired for O-PS synthesis in Salmonella species of the O-antigengroups C1 and L (Kuhn et al., FEMS Microbiol. Rev. 54 (1988), 195-222;Mäkelä et al., J. Gen. Microbiol. 60 (1970), 91-106), as well as in someserotypes of E. coli and in S. dysenteriae type 1 (Kuhn et al., FEMSMicrobiol. Rev. 54 (1988), 195-222; Schmidt et al., J. Bacteriol. 127(1976), 755-762; Klena and Schnaitman, Microbiol. Rev. 57 (1993),655-682). A further gene, termed rfp, encodes a galactosyl transferaseand is necessary for the production of full-length O-PS in S.dysenteriae type 1 (Klena and Schnaitman, Microbiol. Rev. 57 (1993),655-682). In addition, serotype conversion can be accomplished viasubstitution of an O-PS sugar promoted by certain phages lysogenic forSalmonella species and S. flexneri (Clark et al. , Gene 107 (1991),43-52; Verma et al. Gene 129 (1993) 101).

In the specific case of V. cholerae, the entire rfb locus ischromosomally encoded. Genes involved in perosamine synthesis (rfbABDE),transport of the polymerized O-PS to the cell surface (rfbGHI), and inthe transfer of tetronic acid onto the perosamine subunit (rfbKLMNO),are sequentially organized to constitute a single operon. In addition,four genes of unknown function, termed rfbPQRS, constitute the 3′ end ofthe operon. Directly adjacent to the rfb operon is the rfbT gene whichdetermines the Inaba and Ogawa serospecificity of 01 strains of V.cholerae. It was recently determined that the Inaba serotype strains,are rfbT mutants (Manning et al., p.77-94. In Vibrio cholerae andCholera: molecular to global perspectives (1994). Wachsmuth, K., Blake,P. A., and Olsvik, φ. (eds.). Washington, D.C.:American Society forMicrobiology.

As noted above, the induction of a local intestinal immune response maybe the most efficient means by which to prevent infection with a numberof enteric pathogens. A proven and effective method by which toaccomplish this is through the use of live oral attenuated vaccinestrains. Vaccine strains such as S. typhi Ty21a and V. choleraeCVD103-HgR noted above undergo an abortive infectious process therebyinducing an immune response closely resembling that effected by naturalinfection. The above two strains possess the distinct advantage of beingextremely safe in humans (Levine et al., Rev. Infect. Dis. 11 (1989),(Suppl 3), 552-567; Cryz et al., Infect. Immun. 61 (1993), 1149-1151;Levine and Kaper, Vaccine 11 (1993), 207-212).

Safety has been found to be the most difficult attribute to achieve inthe development of live oral vaccine strains. Most often, candidatevaccine strains either induce a protective immune response but with anunacceptable rate of adverse reactions or are safe but non-protective(Lindberg, In Vaccine and Immunotherapy. Cryz Jr,S. J. (ed.). New York:Pergamon Press Inc. (1991), pp. 95-112; Levine and Hone, In Vaccine andImmunotherapy. Cryz Jr,S. J. (ed.). New York: Pergamon Press Inc.(1991), pp. 59-72).

Given the above, it is desirable to utilize approved live oralattenuated vaccine strains as carriers for the delivery of heterologousvaccine antigens to the intestinal tract. Attempts to utilize the S.typhi Ty21a strain as a carrier for vaccine antigens has not yieldedpromising results (Curtiss III, In: New generation vaccines. Woodrow, G.C. and Levine, M. M. (eds.) New York: Marcel Dekker Inc. (1990), pp.161-188; Cardenas and Clements, Clin. Microbiol. Rev. 5 (1992),328-342). This in large part can be accounted for by the fact that thisstrain was developed using a potent chemical mutagen which inducedmultiple mutations. Therefore, the precise attenuating mutation isunknown. Furthermore, the Ty21a strain replicates poorly in vivorequiring multiple doses of vaccine to be administered. In contrast, theCVD103-HgR vaccine strain was constructed using recombinant DNAtechnology allowing for the precise genetic lesions to be identified(Ketley et al., FEMS Microbiol. Lett. 111 (1993), 15-22). Furthermore,this strain appears to replicate well in vivo as evidenced by the factthat only a single dose of vaccine is required to induce a high level ofimmunity against experimental cholera (Levine et al., Lancet ii (1988),467-470).

Initial attempts to utilize the above strains as carriers envisioned thedevelopment of bivalent vaccines. In such a case, the recombinant strainwould co-express two O-PS antigens. However, the successful developmentof such bivalent vaccine strains has proven to be extremely difficultfor a variety of reasons, some of which are just becoming apparent.First, experimental data has shown that covalent linkage between theO-PS moiety and LPS core region appears to be a prerequisite for theefficient induction of immunity (Beckmann et al., Nature 201 (1964),1298-1301; Kuhn et al., FEMS Microbiol. Rev. 54 (1988), 195-222;Attridge et al., Microb. Path. 8 (1990), 177-188; Baron et al., Infect.Immun. 55 (1987), 2797-2801). Second, the co-expression of two O-PSentities often results in the masking of one antigen thereby bluntingthe immune response (Attridge et al., Microb. Path. 8 (1990), 177-188;Forrest et al., Vaccine 9 (1991), 515-520). Third, the recombinantstrain must still fully express the protective antigens associated withthe carrier strain. Finally, expression of the foreign antigen shouldnot adversely affect the ability of the bivalent strain to eitherreplicate in vivo or colonize the mucosal surfaces.

The following examples illustrate the practical problems encountered inthe construction of bivalent vaccine strains. Formal et al. (Infect.Immun. 34 (1981), 746-750) have introduced the 120 Mdal virulenceplasmid of S. sonnei into S. typhi Ty21a via conjugation. The resultinghybrid strain, termed 5076-1C, expressed the O-PS antigen of S. sonneiencoded by the plasmid on the surface of Ty21a as a capsular-likematerial unbound to S. typhi LPS core (Seid et al., J. Biol. Chem. 259(1984), 9028-9034). Immunization of volunteers with this strain resultedin a vigorous anti-S. sonnei LPS antibody response. However, inchallenge studies, various lots of this vaccine were unable toconsistently afford significant protection against S. sonnei disease(Herrington et al., Vaccine 8 (1990), 353-357; Black et al., J. Infect.Dis. 155 (1987), 1260-1265; Van De Verg et al., Infect. Immun. 58(1990), 2002-2004). The precise reason for this variable protection hasnot been identified. Possible explanations include, 1) the presence ofthe S. sonnei antigen on the surface of the Ty21a strain interfered withits ability to effectively colonize, 2) the virulence plasmid was shownto be genetically unstable within Ty21a giving rise to spontaneousdeletions which interfered with the expression of the S. sonnei O-PS andother virulence-associated antigens, 3) expression of the S. sonneiplasmid in Ty21a could have led to a deleterious effect manifested onlyin vivo such as reduced survival, multiplication or colonization.

A bivalent vaccine strain was constructed by introducing the genesencoding for V. cholerae O-PS biosynthesis into Ty21a yielding strainEX645. This strain induced a modest anti-V. cholerae LPS immune responsewhen fed to volunteers even though the heterologous O-PS was coupled tothe LPS core (Forrest et al., J. Infect. Dis. 159 (1989), 145-146). Onlya modest level of protection was afforded against experimental cholerafollowing immunization with EX645. Subsequent studies showed that thelonger S. typhi O-PS probably masked the somewhat shorter V. choleraeO-PS units accounting for the poor immune response. A derivative ofEX645, termed EX880, was developed by inactivating genes involved in theexpression of the S. typhi O-PS. EX880 was found to induce a far morevigorous anti-V. cholerae LPS antibody response compared to EX645(Attridge et al., Infect. Immun. 59 (1991), 2279-2284). The anti-S.typhi LPS response was minimal.

The rfb/rfc and the rfa_(R1) loci of S. sonnei were introduced intoCVD103-HgR by the use of compatible plasmids (Viret et al., Mol.Microbiol. 7 (1993), 239-252). This allowed for the efficient expressionof the S. sonnei O-PS coupled to LPS core. However, when these samegenetic loci were introduced into the chromosome of CVD103-HgR (strainsCH3 and CH9), little if any S. sonnei O-PS was covalently coupled to LPScore (Viret and Favre, Biologicals 22 (1994), 361-372). Instead, thematerial was expressed on the surface of CVD103-HgR as a capsular-likematerial.

The above observations suggest the following, 1) heterologous O-PS canbe efficiently coupled to homologous or heterologous LPS core only ifthe synthesis of homologous O-PS is suppressed, 2) under appropriateconditions it may be possible to covalently couple heterologous O-PS tothe unique core of V. cholerae thereby obviating the need forintroducing genes coding for a heterologous core molecule, and 3) theco-expression of two distinct O-PS molecules by the same carrier strainresulting in a bivalent vaccine may not be feasible. Thus, the efficientsimultaneous expression of two complete LPS molecules each presentingdifferent O-PS moieties may be beyond the capacity of a single hoststrain. Possible reasons include interference with the expression of therespective genes at the transcriptional level, competition for limitingcomponents involved in the biosynthesis of the outer membrane structure,such as molecules involved in the transposition of the O-PS molecule tothe outer surface of the cell, or competition between the O-PS moleculesfor transfer or binding to available sites on the LPS core molecule.

In an attempt to circumvent these problems previously spontaneous,undefined mutants of V. cholerae CVD103-HgR which are deficient in thesynthesis of O-PS were isolated. Such strains were capable of supportingthe covalent attachment of S. sonnei O-PS encoded by the chromosomallyintegrated rfb/rfc loci to an LPS core. However, the undefined nature ofthe mutation(s), present in such strains render them unacceptable forhuman use.

SUMMARY OF THE INVENTION

Thus, the technical problem underlying the present invention is toprovide live attenuated vaccine carrier strains, which are useful forthe expression and delivery of heterologous O-antigen (O-PS) fromgram-negative bacteria in such a way that the heterologous O-PS caninduce an immune response and which are safe and acceptable foradministration as a vaccine.

The solution to said technical problem is achieved by providing theembodiments characterized in the claims. It has been surprisingly foundthat a defined genetic modification can be introduced in a liveattenuated vaccine strain, which does not interfere with the functionsof the carrier strain required in order to make said strain suitable ascarrier for a heterologous antigen, and which leads to a deficiency ofsaid strain in the synthesis of homologous O-PS, thereby allowing toefficiently express a desired heterologous O-PS in such a manner thatthe heterologous O-PS is covalently coupled to the LPS core and caninduce an immune response.

The embodiments of the present invention inter alia allow for theconstruction of monovalent vaccine strains with the followingcharacteristics, 1) use of a live oral attenuated vaccine strain,preferably V. cholerae CVD103-HgR, suitable for human use as a carrierfor heterologous antigens, 2) modification of said carrier strain so asto render it deficient in the synthesis of homologous O-PS byintroduction of precise mutations, e.g. within the rfb gene which arenon-lethal, halt the synthesis of homologous Inaba O-PS and allow forthe expression and covalent coupling of heterologous O-PS to the LPScore, 3) containing genes necessary for the production of heterologous,polymerized LPS molecules derived from other enteric pathogens andexpressing them, wherein stable expression is achieved by integration ofthe cloned heterologous genes at a site which does not adversely affectthe phenotype of the carrier strain, specifically, those traits whichwould allow it to induce a protective immune response following oraladministration, 4) expression of the heterologous O-PS genes in such amanner that the encoded O-PS is covalently coupled to either the LPScore of the carrier strain or a heterologous LPS core produced by thecarrier strain following the introduction of the appropriate rfa locus,5) the LPS molecule bearing the heterologous O-PS moiety is expressed onthe surface of the carrier strain, preferably integrated into the outermembrane protein, and 6) the genotype/phenotype of the carrier strainwhich renders it suitable for human use is maintained.

In order to develop such vaccine strains various genetic modificationswere introduced in the genes for expression of the O-PS of the carrierstrain in order to eliminate synthesis of the O-PS.

Surprisingly, the deletion of the entire Inaba rfb locus (about 20 kb)had a lethal effect upon CVD103-HgR and its S. sonnei rfb/rfc-bearingderivatives (strains CH3 and CH9). Therefore, it was assumed that theremust be genes encoding for essential functions within or adjacent to therfb locus and that strains deficient in such functions would be unableto multiply, presumably due to their inability to synthesize afunctioning outer membrane structure. It was therefore sought tointroduce specific deletions, for example, within three distinct regionsof the rfb locus. The goal was to try to introduce non-lethal deletionsinto the rfb locus which would, in addition to halting expression of thehomologous Inaba O-PS moiety, support the covalent coupling of theheterologous S. sonnei O-PS to the Inaba LPS core. The first suchconstruct was a rfbEGHI mutant. The rfbE locus encodes the perosaminesynthetase whereas the rfbG, H, and I loci are involved in the transportof the Inaba O-PS through the outer membrane (Manning et al. p.77-94. InVibrio cholerae and Cholera: molecular to global perspectives (1994).Wachsmuth K., Blake, P. A., and Olsvik φ. (eds.). Washington,D.C.:American Society for Microbiology). The rfbEGHI deletion was foundto be lethal in CVD103-HgR. In a second strain, deletion of the rfbNlocus (which is involved in the synthesis of the perosamine substituent3-deoxy-L-glycero-tetronic acid), unexpectedly resulted in only weakproduction of the heterologous S. sonnei O-PS which was unbound to theInaba core. Therefore specific gene functions within the Inaba rfb locusare useful for both the expression of the heterologous S. sonnei rfbgenes and its covalent coupling to the V. cholerae LPS core in as of yetunidentified manner. Next the rfbA and rfbB loci were inactivated bydeleting a 1.2 kb fragment overlapping the junction between the twoloci. These loci are involved in the synthesis of the perosaminecomponent of the Inaba O-PS. Specifically, the RfbA protein isassociated with enzymes having phospho-mannose isomerase ormannose-1-phosphate guanyl transferase activity while the rfbB lociencodes a putative phospho-manno mutase. The introduction of therfbA/rfbB mutation into CVD103-HgR containing the S. sonnei rfb/rfc lociallowed for the expression and covalent coupling of the S. sonnei O-PSto the Inaba LPS core giving rise to full length hybrid LPS molecules.Recombinant strains expressing the Inaba rfbA/rfbB deletion togetherwith the S. sonnei rfb/rfc loci with or without the R1 core were foundto be genotypically and phenotypically stable upon passage in vitro.Furthermore, these strains possessed all the characteristics of theCVD103-HgR strain which render it suitable for human use, including, 1)lack of cholera toxin activity, 2) production of non-toxic B subunit ofcholera toxin, 3) expression of toxin co-regulated pili, and 4) theability to grow in the presence of elevated levels of Mercury ions.

Accordingly, the present invention relates to live attenuated vaccinestrain against gram-negative enteric pathogens characterized by thefollowing properties:

(a) deficiency to express homologous O-PS due to a, defined geneticmodification, and

(b) capability to efficiently express heterologous O-PS in such a mannerthat said heterologous O-PS is covalently coupled to the LPS core.

As used herein, the term “defined genetic modification” encompasses anymodification(s) which has (have) been introduced by recombinant DNAtechniques and which is (are), in contrast to modifications introducedby random mutagenesis or due to spontaneous mutations, defined withrespect to its nature and location. Said modifications can be deletions,additions, substitutions or rearrangements of nucleotides, but shouldpreferably not give raise to the occurrence of revertants. Suitablegenetic modifications in accordance with the present invention can beintroduced by the person skilled in the art following the teaching givenin the Examples below. Such modifications should not interfere with thefunctions of the carrier strain required in order to make said strainsuitable as carrier for a heterologous O-PS, but should sufficientlyeliminate the expression of homologous O-PS. For example, saidmodifications affecting the biosynthesis of the homologous O-PS shouldnot adversely affect the expression of genes which are essential for thesynthesis of complete LPS comprised of heterologous O-PS, e.g. the genesinvolved in the synthesis of lipid A, the LPS core, the synthesis andtransport of O-PS to the outer cell surface and anchoring the LPSmolecules into the outer membrane.

Due to said modifications said strains synthesize LPS molecules whichonly consist of the homologous lipid A and homologous and/or, in aspecific embodiment which is described below, a heterologous LSP core.Preferably, said modifications are deletions. As used herein, the term“deficiency to express homologous O-PS” means that the expression of thehomologous O-PS is entirely eliminated or at least reduced such that theefficient expression of the desired heterologous O-PS, and its covalentbinding to the LPS core of the carrier strain or, alternatively, to aheterologous LPS core is made possible.

As used herein, the term “capability to efficiently express heterologousO-PS” means the capability to express said O-PS in such a way, that theamounts of heterologous O-PS produced are sufficient to elicit an immuneresponse.

In a preferred embodiment, the vaccine strain carries a defined geneticmodification within the genes involved in O-PS biosynthesis contained inthe rfb, rfc, and/or rfp loci or any combination thereof.

In a particularly preferred embodiment, the vaccine strain carries adefined genetic modification within the rfbA-, rfbB-, rfbD and/orrfbE-gene or any combination thereof, preferably within the rfbA- and/orrfbB-gene.

Most preferred is a vaccine strain, wherein said genetic modification isa deletion corresponding to the deletion shown for pSSVI255-20 (DSMdepository number DSM13426) in FIG. 1. This deletion is located at thebeginning of the rfb_(inaba) operon and concerns the elimination of a1.2 kbp HindIII fragment. It inactivates the rfbA- and rfbB-genes whichare involved in the biosynthesis of the perosamine O-antigen subunit.

Suitable vaccine strains can be selected by the person skilled in theart, depending on the desired purpose. Such strains are, for example,CH19 (DSM depository number DSM13420), CH21 (DSM depository numberDSM13421), CH22 (DSM depository number DSM13422), CH24 (DSM depositorynumber DSM13423), CH25 (DSM depository number DSM13424) or CH30 (DSMdepository number DSM13425), described below.

In a preferred embodiment, said vaccine strain is an E. coli strain, astrain of the genus Shigella, S. typhi, O1 or O139 V. cholerae,Heliobacter pylori or Campylobacter jejuni. Preferred S. typhi strainsare S. typhi Ty21a, S. typhi CVD908, or S. typhi CVD908 containingadditional attenuating mutations. Examples of additional attenuatingmutations are mutations in the viaB or htpR genes encodingtranscriptional signals such as the RpoS sigma factor or in genesinvolved in virulence traits such as the resistance to environmentalstress or the capacity to adapt to new growth conditions or in genesinvolved in the synthesis of aromatic acids.

Preferred V. cholerae strains are V. cholerae CVD103-HgR, V. choleraeCVD103-HgR, CVD110, CVD111, CVD112, Bengal-15 or Peru-14.

Preferred Shigella strains are S. dysenteriae, S. sonnei, S. boydii, orS. flexneri serotype Y.

The above vaccine strains can be used for the efficient expression ofheterologous O-PS. For this purpose a heterologous gene or a set ofheterologous genes coding for O-PS are inserted into the vaccine strainby methods known to the person skilled in the art, for example bymethods described in the Examples, below.

Accordingly, the present invention relates to vaccine strains furthercharacterized by the presence of a heterologous gene or a set ofheterologous genes coding for O-PS.

The insertion of said gene(s) encoding a heterologous O-PS should becarried out in such a manner that (i) said gene(s) are stably expressedand allow for the synthesis of complete full-length, smooth LPSessentially indistinguishable from the parent strain, and (ii) an intacthybrid LSP is formed composed of the lipid A of the vaccine straincoupled to the homologous core region. Thus, when inserting said gene(s)the person skilled in the art should

i) use a bacterial carrier strain devoid of the genes coding for thehomologous O-PS,

ii) use a plasmid, for example pMAK700oriT, composed of all the genescoding for the heterologous O-PS, flanked by homologous genetic regionscorresponding to the locus where the said heterologous O-PS genes are tobe inserted, and

iii) then proceed as described in Example 3, below.

In a preferred embodiment of the vaccine strains, the heterologousgene(s) is (are) present either on a plasmid vector or stably integratedinto the chromosome of said strain at a defined integration site whichis to be non-essential for inducing a protective immune response by thecarrier strain.

The set of heterologous genes should be cloned in a deletion vectorcomposed of a thermosensitive replicon, for example, pMAK700oriT and ahomologous genetic region corresponding to the gene where the insertionis to take place. The heterologous genes will be cloned in the middle ofthe homologous region. For the integration of the heterologous genesthis plasmid should be introduced into a suitable carrier strain andthereafter handled like in Examples 5, 6, 7 and 8, below.

Suitable sites for integration of the heterologous gene(s) into thechromosome of the vaccine strain are genes which in no way will effectproperties of the strain necessary for its immunogenecity and safety.

In a preferred embodiment, said heterologous gene or set of heterologousgenes are integrated into either the hlyA, hlyB, rfbA, and/or rfbA/rfbBloci of V. cholerae.

A further particular preferred embodiment relates to a S. typhi strain,wherein said heterologous gene or set of heterologous genes areintegrated into either the H₂S production gene, ilv, viab, htpR genesencoding transcriptional signals such as the RpoS sigma factor, genesinvolved in virulence traits such as the resistance to environmentalstress or the capacity to adapt to new growth conditions, or any geneinvolved in the synthesis of aromatic acids. Genes involved in theresistance to environmental stress or the capacity to adapt to newgrowth conditions are genes of the OmpR-EnrZ system, PhoP-PhoQ systemand cya-crp transcription regulation system. Genes involved in thesynthesis of aromatic acids are, for example, aroA, aroC and aroD.

Alternatively, the above vaccine strains contain the rfa, rfe, rfp,and/or any additional gene(s) necessary for the synthesis of completesmooth heterologous LPS which are integrated in tandem into a singlechromosomal site or independently integrated into individual sites.

Additional genes necessary for the synthesis of complete smoothheterologous LPS are for example, rfc and rff. Integration of the abovegenes in such a way that they are correctly and in a coordinate mannerexpressed can be carried out by the person skilled in the art accordingto well known methods or, for example, described in Hamilton et al., J.Bacteriology 171 (1989), 4617-4622.

Such vaccine strains allow expression of heterologous O-PS which iscovalently coupled to a heterologous LPS core region, which, preferably,exhibits a degree of polymerization essentially indistinguishable fromthat of native LPS produced by the enteric pathogen. Such vaccinestrains can, if desired, modified in such a way that they are deficientin the synthesis of homologous LPS core.

In a preferred embodiment, the heterologous rfa genes encode the Ra, R1,R2, R3, R4, K-12 or B LPS core, preferably the R1 core.

The invention also relates to a live vaccine comprising the abovevaccine strain and optionally a pharmaceutically acceptable carrierand/or a buffer for neutralizing gastric acidity and/or a system fordelivering said vaccine in a viable state to the intestinal tract.

Said vaccine comprises an immunoprotective and non-toxic amount of saidvaccine strain. Suitable amounts can be determined by the person skilledin the art and are typically 10⁷ to 10⁹ bacteria.

Pharmaceutically acceptable carriers, suitable neutralizing buffers, andsuitable delivering systems can be selected by the person skilled in theart.

In a preferred embodiment said live vaccine is used for immunizationagainst gram-negative enteric pathogens.

The mode of administration of the vaccines of the present invention maybe any suitable route which delivers an immunoprotective amount of thevaccine to the subject. However, the vaccine is preferably administeredorally or intranasally.

The invention also relates to the use of the above vaccine strains forthe preparation of a live vaccine for immunization against gram-negativeenteric pathogens. For such use the vaccine strains are combined withthe carriers, buffers and/or delivery systems described above.

The following examples illustrate the invention.

In summary, the utility of Inaba rfbA/rfbB deletion mutants as carriersor vectors for heterologous O-PS antigens is illustrated. The rfb locusof O139 V. cholerae was cloned on a about 32 kb fragment and integratedinto the hlyA::mer locus of the rfbA/rfbB deletion mutant. Thisconstruct expressed O139 O-PS which was coupled to the Inaba core andrecognized by specific anti-O139 antibodies. Similarly, the rfb/rfp locifrom S. dysenteriae which allow the production of O-PS were cloned on a13.8 kb fragment and integrated into the rfbA/rfbB deletion mutant ofCVD103-HgR as described above. In this construct the S. dysenteriae O-PSwas produced on the cell surface, covalently coupled to the core andrecognized by specific anti-S. dysenteriae O-PS. However, this constructexpressed only very short LPS molecules instead of the full ladder-likestructure associated with native S. dysenteriae LPS. However, theaddition of the rfe gene from E. coli, believed to be involved in thepolymerization of O-PS, on a plasmid or integrated into the chromosomeof the construct, resulted in the synthesis of a LPS with a phenotypeindistinguishable from that of native S. dysenteriae.

EXAMPLE 1 Cloning and Physical Mapping of the rfb Locus From V. choleraeCVD103-HgR

Preparation of the gene bank. A V. cholerae CVD103-HgR DNA gene bank wasprepared in the low-copy number cosmid pLAFR5 (Keen et al., Gene 70(1988), 191-197). DNA fragments from isolated CVD103-HgR chromosomal DNAwere generated by partial Sau3A restriction and size fractionated on asucrose gradient. Fractions containing 20 to 30 kb fragments werepurified and ligated to the BamHI and ScaI-cut vector. The ligatedmixture was packaged in vitro (Gigapack II Plus packaging kit,Stratagene GMBH, Zürich, Switzerland) according to the manufacturer'sinstructions. The packaged DNA was then transfected into E. coli strainHB101 and the resulting culture was plated out onto LB plates containing12.5 μg/ml tetracycline (LBTc plates) to select for transfectants.Resistant colonies were pooled, aliquoted and the aliquots were storedin 40% glycerol at −70° C.

Screening of the gene bank. One frozen aliquot of the cosmid bank wasdiluted and plated out on LBTc plates. Arising colonies were transferredonto nitrocellulose filters. Filters were then processed forimmunodetection according to published protocols (Sambrook et al.,Molecular cloning, 2nd edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor USA, (1989)). Probing the filters with theInaba/Ogawa specific monoclonal antibody (mAb) VCO4 (previously calledH4; Gustafsson and Holme, J. Clin. Microbiol. 18 (1983), 480-485)allowed the isolation of several independent clones, which remainedstrongly positive when retested with the same mAb. Three clones namedpSSVI255-3, pSSVI255-5, and pSSVI255-7 were further characterized.

Restriction analysis of pSSVI255-3, pSSVI255-5 and pSSVI255-7. Therestriction pattern obtained with a variety of restriction enzymesindicated a large degree of overlap among the three clones. All threeclones were mapped using EcoRI, SacI, and PstI. With the aid of a knownDNA sequence of an approximately 20 kb SacI fragment encompassing therfb locus from the El Tor Ogawa V. cholerae strain O17 (Manning et al.,p.77-94. In Vibrio cholerae and Cholera: molecular to globalperspectives (1994). Wachsmuth K., Blake, P. A., and Olsvik Y. (eds.).Washington, D.C.:American Society for Microbiology, the exact locationof the rfb locus in each clone could be determined. On the basis of thisinformation, clone pSSVI255-7 (FIG. 1) was exclusively used for furtherwork.

EXAMPLE 2 Construction of Deletion Plasmids for the Introduction ofChromosomal Deletions in V. cholerae

In order to maximize the probability of obtaining a successful deletionwithin the rfb locus of V. cholera, four distinct fragments within therfb locus were selected to be individually deleted. FIG. 1 summarizesthe various deletion vectors that were generated. Plasmids pSSVI205-1and pSSVI205-2 were constructed by removing 23.5 kb of DNA between theouter SacI sites in pSSVI255-7, and subcloning, in both orientations,the remaining insert into the blunted HindIII site of prvAK700oriT (FIG.2). The latter plasmid corresponds to the suicide vector pMAK700(Hamilton et al., J. Bacteriol. 171 (1989), 4617-4622) made mobilizableby the addition of the oriT region from plasmid pJFF350 (Fellay et al.,Gene 76 (1989), 215-226). Plasmid pSSVI255-12 corresponds to pMAK700oriTbearing the 10.5 kb SalI-HindIII central fragment of pSSVI255-7 fromwhich the 3.8 kb internal BamHI fragment was deleted. This deletioninactivates the rfbEGHI genes. Gene rfbE is the putative perosaminesynthetase, whereas rfbG, H and I are involved in the transport of therfb_(inaba)-encoded O-PS components through the outer membrane (Manninget al., p.77-94. In Vibrio cholerae and Cholera: molecular to globalperspectives (1994). Wachsmuth K., Blake, P. A., and Olsvik φ. (eds.).Washington, D.C.:American Society for Microbiology. Plasmids pSSVI255-19and pSSVI255-20 were both derived from pSSVI25S-7 through subcloning ofdefined restriction fragments into the high-copy-number vector pMTL22p(Chambers et al., Gene, 1988, 68:139-149), deletion of a central region,and further. subcloning of the resulting insert into pMAK700oriT.pSSVI255-19 corresponds to the 5.3 kb ClaI fragment (Map position 15770to 21030, FIG. 1) from which the internal 1.9 kb SalI fragment wasdeleted. The deletion overlaps the, rfbN gene which is believed to beinvolved in the synthesis of the perosamine substituent3-deoxy-L-glycero-tetronic acid. pSSVI255-20 corresponds to the 5.3 kbBamHI-SacI fragment located at the beginning of the rfb_(Inaba) operon(Map position 18500 to 23400), from which the 1.2 kb HindIII internalfragment was deleted. This deletion inactivates the rfbA and rfbB geneswhich are directly involved in biosynthesis of the perosamine O-antigensubunit. RfbA function is closely associated to proteins withphospho-mannose isomerase or mannose-1-phosphate guanyl transferaseactivity and RfbB is a putative phospho-manno mutase.

EXAMPLE 3 Introduction of rfbAB Deletions in Target Carrier Strains:Construction of Strains CH15, CH19 and CH30

The various plasmids described in Example 2 were first transferred byelectroporation into the E. coli mobilization strain S17.1 (Simon et al.Bio/Technology 1 (1983), 784-791) and then mobilized into V. choleraeCVD103-HgR. In addition, pSSVI255-20 was mobilized into the El Tor Ogawavaccine strain CVD111.

Transconjugants were isolated by plating at 30° C. on selective BHI-Cmplates. The transconjugants were then propagated at 30° C. in liquidcultures (BHI-Cm medium) and suitable dilutions were plated on BHI-Cmplates and incubated at 30° C. or 41-42° C. Typically, the platingefficiency at 41-42° C. was about 10⁴-fold lower than at 30° C. Since,by virtue of its thermosensitive replicon, the plasmid is unable toreplicate at 41° C. or more, colonies at the non-permissive temperatureshould arise from those rare cells carrying the entire deletion plasmidin, their chromosome. A series of colonies capable of growth at 41-42°C. were further streaked onto BHI-Cm plates incubated at 41-42° C. Theselection of cells free of vector sequences was performed via streakingon non-selective BHI plates incubated at 30° C. Subsequently,immunological screening allowed for the isolation of colonies whichresponded negative for the VCO4 mAb. This antibody recognizes both theOgawa and Inaba O-PS. These colonies were further screened to confirmthat they had lost the chloramphenicol resistance trait inherent to thevector.

Such stable integrants were however not always isolated, indicating thatsome deletions were lethal. Thus, implementation of strains in whicheither the entire rfb locus or the rfbEGHI genes were deleted could notbe obtained. The strain obtained by introduction of the rfbN deletion inCVD103-HgR using pSSVI255-19 was designated CH15, and rfbAB deletionmutants in CVD103-HgR and CVD111 using pSSVI255-20 were named CH19 andCH30, respectively. These deletion strains were geneticallycharacterized by Southern hybridization using probes specific for therfb locus. The genetic structure of all strains tested was found to beconform to expectations.

EXAMPLE 4 Introduction of rfbAB Deletions in Recombinant V. choleraeStrains Expressing S. sonnei O-PS Alone or in Combination With the E.coli rfaR1 LPS Core. Construction of Strains CH13, CH14, CH17, CH21

The above described plasmids were mobilized into strains CH3 and CH9 asdescribed in Example 3.

As was the case for CVD103-HgR, introduction of the entire rfb locusdeletion in CH3 or CH9 could not be achieved, presumably due to itslethal effect. Likewise, introduction of the 2 kb SalI deletion frompSSVI255-19 or the 1.2 kb HindIII deletion from pSSVI255-20 were notsuccessful in strain CH3. The rfbEGHI deletion mutants arising from theintegration of pSSVI255-12 into CH3 and CH9 are referred to as CH13 andCH14, respectively. Insertion of the rfbN deletion in CH9 usingpSSVI255-19 was designated CH17, and a CH9 deletion mutant carrying therfbAB deletion from pSSVI255-20 was named CH21. These deletion mutantswere genetically characterized by Southern hybridization using probesspecific for either the rfb_(Inaba) or rfb/rfc_(sonnei) loci in additionto a probe for the hlyA gene, the integration target for therfb/rfc_(sonnei) locus. The genotype of all strains tested was found tobe conform to expectations.

EXAMPLE 5 Integration of the rfb/rfc_(sonnei) Locus Into CH19.Construction of Strain CH22

Since we could not produce a CH3 deletion mutant using pSSVI255-20, agenotypically similar strain, CH22, was constructed by the reverseapproach, namely the integration of rfb/rfc_(sonnei) genes carried onplasmid pSSVI201-1 (FIG. 3) into the chromosome of CH19. pSSVI201-1 wasinitially used for the construction of strain CH9. The plasmid wasmobilized from the E. coli strain S17.1 (pSSVI201-1) into CH19. A poolof transconjugants was then submitted to the integration procedureexactly as described in Example 2, except that the presence of theintact rfb/rfc_(sonnei) locus was checked at each step of the procedureby immunological screening using mAB Sh5S (Viret et al., Infect. Immun.60 (1992), 2741-2747). A stable Cm^(s)/Sh5S+ integrant was isolated andnamed CH22 (FIG. 4).

EXAMPLE 6 Integration of the rfb Locus From V. cholerae O139 Strain MO45Into CH19: Construction of Strain CH25

Construction and screening of a DNA gene bank. A chromosomal gene bankderived from the wild type V. cholerae O139 strain MO45, the referenceO139 epidemic strain, was constructed in pLAFR5 following the sameprocedure than that described in Example 1. The bank was thenimmunologically screened using an MO45-specific rabbit polyclonalantibody. A total of 13 cosmid clones were isolated which stronglyreacted with the polyclonal antibody. These clones were then submittedto restriction analysis using a variety of restriction enzymes in orderto determine the level of overlapping.

LPS expression in E. coli. LPS small-scale preparations (minipreps) weremade from strains selected on the basis of the restriction pattern ofthe plasmids. Aliquots from these minipreps, together with LPS miniprepsfrom the negative controls CH19 and HB101 (pSSVI212-15) and the positivecontrol MO45, were then analyzed by silver stained SDS-PAGE andimmunoblotting (Western blot) using as primary antibody the anti-O139polyclonal serum described above. The developing antibody was ahorseradish peroxydase-conjugated goat anti-rabbit IgG (BoehringerMannheim AG, Rotkreuz, Switzerland). Procedures for blotting of the gelonto a nitrocellulose membrane, subsequent incubation with antibodiesand detection were as previously described (Viret et al., Infect. Immun.60 (1992), 2741-2747).

Results, shown in FIG. 5, indicate that most of the clones displayed aLPS pattern identical to that of MO45 (lane 4, characteristic of allO139 strains) in the low size range (lanes 6-11). However, only oneclone, namely pSSVI212-3 (lane 5), was identical to the full MO45 LPSpattern, i.e., with both low and high molecular weight material, thelatter being typical of capsular polysaccharides. The slight unspecificresponse from the CH19 carrier strain (lanes 2, 9-11) may be due to somecommon epitopes in the LPS core of O1 and O139 strains. Since capsularpolysaccharides are considered necessary to produce a meaningful immuneresponse against O139 pathogens, pSSVI212-3 was chosen for further work.

Construction of CH25. Further restriction analysis of pSSVI212-3,indicated that no NotI restriction site occurred within the about 30 kbinsert. However, NotI sites were available within the cosmid vectorpLAFR5 on either side of the insert at 1.0-1.5 kb from the cloning site.Accordingly, the about 32 kb NotI fragment containing the O139 rfb locuswas subcloned blunt into the SalI site of integration vector pSSVI209(FIG. 6) to produce pSSVI220. Plasmid pSSVI220 was then electroporatedinto E. coli S17.1 and mobilized into CH19. The integration procedure ofthe O139 rfb locus at the hlyA::mer locus was as described in Example 4,except that the presence of the intact O139 rfb locus was checked usingthe anti-O139 polyclonal antiserum described above which had beenpreadsorbed against CH19. Several colonies grown at 30° C. withoutantibiotic selection were found which were Cm^(s) and reactive with theanti-O139 antibody. One of these colonies was kept and the strain wasnamed CH25.

EXAMPLE 7 Integration of the S. dysenteriae rfb/rfp Locus Into theChromosome of CH19: Construction of Strain CH23

Construction of integration plasmid pSSVI208-2. The source of therfb/rfp locus from S. dysenteriae 1 was plasmid pSS37 (Sturm et al.,Microb. Path. 1 (1986), 289-297). The XbaI-EcoRV insert from pSS37 wasfirst cloned in tandem with the Sce-Km cassette (Viret, BioTechniques 14(1993), 325-326) into the SalI site of the low-copy number vector pGB2(Churchward et al., Gene 31 (1984), 165-171) to give pSS37-1K. The 13.8kb insert was then excised with SalI and cloned into the SalI site ofthe integration vector pSSVI199S (FIG. 7) in both orientations toproduce pSSVI208-1K and pSSVI208-2K. The SceI-Km cassette frompSSVI208-2K was then excised by SceI restriction and self-ligation ofthe plasmid to yield pSSVI208-2 (FIG. 8).

Construction of CH23. Plasmid pSSVI208-2 was electroporated into E. coliS17.1, mobilized into CH19, and transconjugants were selected on LB-Cmplates at 30° C. The subsequent integration was as described in Example4 except that a polyclonal anti-S. dysenteriae 1 rabbit antiserum wasused for the screening of colonies containing the rfb/rfp locus. Severalcolonies grown at 30° C. without antibiotic selection were found whichwere Cm^(s) and reactive with the anti-S. dysenteriae 1 antibody. Qne ofthese colonies was named CH23.

EXAMPLE 8 Integration of the rfe Gene From E. coli Into CH23.Construction of Strain CH24

Rationale for the use of the rfe gene. Previous experimentation hadshown that expression of the rfb/rfp locus from S. dysenteriae 1 into V.cholerae does not result in the production of a complete LPS ladder asseen with the native S. dysenteriae 1 LPS. However, it could bedemonstrated that co-expression of the E. coli rfe gene, which encodesthe enzyme UDP-N-acetyl-glucosamine::undecaprenylphosphateN-acetylglucosamine-1-phosphate transferase (Meier-Dieter et al.,J.Biol.Chem., 267. (1992), 746-753), together with the S. dysenteriaerfb/rfp locus allowed the defect to be overcome, resulting in theproduction of an LPS ladder indistinguishable from that of S.dysenteriae 1.

Construction of integration plasmid pSSVI219. Accordingly, a plasmid forthe integration of the rfe gene into the chromosome of CH23 wasconstructed. The 1.5 kb XmaIII-ClaI fragment from plasmid pRL100(Meier-Dieter et al., J.Biol.Chem., 267 (1992), 746-753) was subclonedblunt into the Klenow-blunted BamHI site of plasmid pMAK/hlyA to givepSSVI219 (FIG. 9).

Construction of CH24. Plasmid pSSVI219 was electroporated into E. coliS17.1, mobilized into CH23 and transconjugants were selected on LB-Cmplates at 30° C. Subsequent integration procedures were as described inExample 4 except that a S. dysenteriae O-PS specific monoclonal antibody(DysH26, unpublished) was used for the screening of colonies with intactrfe gene. The DysH26 mAb specifically recognizes highly polymerized S.dysenteriae LPS and therefore discriminates between cells containing anactive or an inactive rfe gene. Several colonies grown at 30° C. withoutantibiotic selection were found which were Cm^(s) and positive for mABDysH26. One of these colonies was named CH24.

EXAMPLE 9 Heterolgous O-PS Expression Form Integrated rfb Loci in V.cholerae rfb_(Inaba) Mutants

Expression of S. sonnei rfb/rfc locus alone or in combination with theE. coli rfa_(R1)locus. The expression of S. sonnei and Inaba LPS in CH3and CH9 and their respective Inaba-negative derivatives was examined onsilver-stained SDS-PAGE gels and in immunoblots, using mAb Sh5S (Viretet al., Infect.Immun. 60 (1992), 2741-2747) or VCO4. FIG. 10 depicts theexpression of S. sonnei and Inaba LPS in the various deletion mutantsand their respective parent strains. All rfb_(Inaba) deletions abolishedthe production of Inaba O-PS (Panels A and C, lanes f to l versus lanesb, d, and e). An unexpected finding was that such deletions also affectthe production of the heterologous S. sonnei O-PS to various degrees.Strains CH3 and CH9 which harbour an intact rfb_(inaba) locus (lanes dand e, respectively) both expressed limited amounts of core-bound S.sonnei O-PS (Panel A). When deletions in genes involved in Inaba O-PStransport/perosamine synthesis or tetronate synthesis were introduced inthese strains (CH13/CH14 and CH15, respectively), S. sonnei O-PS waspoorly expressed and remained unbound (lanes f, g and i of Panel B). Incontrast, deletions specific for perosamine synthesis (strains CH21 andCH22) allowed for the expression of large amounts of. core-boundheterologous S. sonnei O-PS, depicted as typical LPS ladder-likestructures in the lower part of the gel (Panel A and B, lanes k and l).

Expression of S. dysenteriae type 1 O-PS in CH23 and CH24. Theexpression of S. dysenteriae LPS in CH23 and CH24 was examined onsilver-stained SDS-PAGE gels and in immunoblots using mAb MASD-1 (Fältand Lindberg, Microb.Path. 16 (1994), 27-41) which recognizes both lowand high molecular weight S. dysenteriae LPS. FIG. 11 clearly shows thatthe expression of complete LPS depends on the presence of the rfe gene(lanes 2,5,6,8,10). Thus, CH24 (lane 10) produces a LPS ladder whichmimics that of the positive controls CH19 and CH3-I⁻ co-infected withpSSV1208-1 and pRL100 (lanes 5 and 6, respectively) and E. coli DH5α(pSS37) (lane 2). In contrast, CH23 (lane 7) synthesizes only a smallamount of low molecular weight material. Strains CH23 (pSSV1219) andCH24 (lanes 8 and 10, respectively) synthesized somewhat less highlypolymerized LPS than their counterparts bearing the rfb/rfp loci on aplasmid (lanes 5 and 6). Therefore, the difference appears to be due tothe lower number of copies of rfb/rfp locus in CH24 versus the strainscarrying the plasmid-borne loci.

Expression of V. cholerae O139 OA. The expression of V. cholerae O139LPS in CH25 was examined in immunoblots using CH19-adsorbed anti-O139polyclonal antiserum. FIG. 12 shows that CH25 (lanes 4 and 5) producesboth low and high molecular weight LPS typical of O139 wild type strainMO45 (lane 3). Comparison of CH25 LPS to LPS from strains in which theO139 rfb locus is carried on low-copy plasmid vectors in E. coli (lanes6 and 7) indicates that the diminution in copy number resulting -fromthe chromosomal integration of the O139 rfb locus in CH25 did not resultin a corresponding reduction in the amount of LPS produced.

EXAMPLE 10 Physiological Characterization of Carrier Strain CH19 andCandidate Vaccine Strains CH21 and CH22

The physiological properties of the genetically defined rfb_(Inaba)deletion mutants cultured at 30° C. are summarized in Table 2. Thephenotype of the deletion mutants was markedly influenced by growth invarious media whereas CVD103-HgR was not. When cultivated at 37° C., allstrains, including CVD103-HgR showed a drastically reduced motility. Theinability of CVD103-HgR to synthesize the Inaba O-PS following theintroduction of the rfbAB deletion (strain CH19) resulted in a phenotypewhich was quite different from the S. sonnei O-PS-expressingcounterparts, CH21, CH22. Thus, CH19 was poorly motile, grew mostly assingle cells or short filaments, and most strikingly, spontaneouslyaggregated in all media tested. Expression of S. sonnei O-PS in therfbAB deletion background (strain CH22) restored many traits expressedby CVD103-HgR such as motility and growth in non-aggregated, mostlynon-filamentous form. Co-expression of the R1 core in strain CH21resulted in filamentous growth and a diminution of motility.

EXAMPLE 11 Further Physiological Characterization of CH21 and CH22

The stability of both strains was studied. A culture of the test strainwas grown to stationary phase at 37° C. in LB medium, diluted 200-foldin the same medium, and further incubated to stationary phase at 37° C.At each round, dilutions of the stationary culture were plated on LBmedium for determination of stability. Genetic stability was defined asthe proportion of colonies still expressing the desired phenotype(expression of S. sonnei O-PS or loss of V. cholera O-PS) after 50 ormore generations of growth. Both strains were found to stably express(>99.9%) S. sonnei O-PS. A similar proportion were found to maintain andexpress the RI LPS core in strain CH21. On the other hand, all testedcolonies failed to express the V. cholerae Inaba O-PS.

Strains CH21 and CH22 were also tested for their innocuity by theY1-adrenal cell assay (Sack and Sack, Infect. Immun. 11 (1975),334-336), for the production of the cholera toxin B-subunit using theGM1 ganglioside-binding assay (Svennerholm and Holmgren, Curr.Microbiol. 1 (1978), 19-23), and for their resistance to mercury. Forthe latter test, cultures of CVD103-HgR, CH21 and CH22 were grownovernight with shaking in BHI medium at 37° C. The stationary phasecultures were diluted either 200-fold in 2 ml BHI containing a series ofHgCl₂ concentrations (BHI/HgCl₂) or 40-fold in 20 ml BHI. The lattercultures were further incubated for 2 hours at 37° C. and again diluted40-fold in 2 ml BHI/HgCl₂ medium containing various HgCl₂concentrations. All cultures were then incubated for up to 3 days at 37°C. with shaking. Positive cultures were recorded by visual examinationon days 1, 2, and 3. In all three assays, CH21 and CH22 wereindistinguishable from CVD103-HgR.

Toxin co-regulated pili, the product of the tcp regulon, is known to bean important factor for V. cholerae adhesion to the intestinal cells. Inorder to evaluate the expression of tcpA, the gene coding for pilin,Western blots of whole-cell extracts of CVD103-HgR, CH21, and CH22, runon SDS-PAGE gels were probed with a pilin-specific antiserum. Resultsshown in FIG. 5 indicate that both CH21 and CH22 produce amounts ofpilin similar to those of CVD103-HgR.

EXAMPLE 12 Immunogenicity of Strain CH22

Sera from mice immunized with killed whole CH22 cells were tested forthe presence of anti-phase I S. sonnei and CVD103-HgR Inaba LPSantibodies. As controls, non-immune sera or sera from mice immunizedwith killed whole CVD103-HgR cells were used. As shown in Table 3,immunization with CH22 induced high titers of anti-S. sonnei LPSantibodies but no anti-Inaba LPS antibodies. In contrast, sera from miceimmunized with CVD103-HgR produced only anti-Inaba LPS antibodies. Serafrom control mice did not react with any of the LPS test antigens.

Legends to the Figures

FIG. 1: Restriction map of the Inaba rfb clone pSSVI255-7 and deriveddeletion vectors.

The arrows depict the direction of transcription of the rfaD gene andrfb_(Inaba) operon. The white boxes delineate the various rfb genes andthe striped boxes denote functional regions. These data are inferredfrom published results (Manning, P. A., et al. p. 77-94. In Vibriocholerae and Cholera: molecular to global perspectives. Wachsmuth K.,Blake, P. A., and Olsvik φ. (eds.). Washington, D.C.:American. Societyfor Microbiology, 1994. The lines below correspond to plasmid insertsindicated on the right. The portions with a thick double line representhomologous regions used for chromosomal integration and excision ofvector sequences. The remaining portions (thin lines) represent thechromosomal regions deleted from each plasmid.

FIG. 2: Restriction map of the mobilizable suicide vector pMAX700oriT.

ori101, pSC101 origin of replication; rep101, gene for thetemperature-sensitive replication initiation protein; cam,chloramphenicol resistance gene; oriT, RP4/RK2 origin of transfer.Coordinates are in base pairs.

FIG. 3: Restriction map of rfb/rfc_(sonnei) locus intgration plasmidpSSVI201-1.

The arrows depict the direction of transcription of the indicated genes.The white box represents the pMAK700oriT vector. The interrupted stripedbox on the map line represents the S. sonnei rfb/rfc locus. Theinterruption denotes that its actual size is larger than represented.The thin lines are the regions homologous to CVD103-HgR chromosomal DNA.hlyA, 5′-end of the hlyA gene; mer, mercury resistance operon; cat,chloramphenicol resistance gene; rep101ts, gene for thetemperature-sensitive replication initiation protein; oriT, RP4/RK2origin of transfer.

FIG. 4: Genetic structure of CH22 at the hlyA::rfb_(sonnei) locus.

The upper map depicts the structure of the hlyA:mer locus in CH19, i.e.,before integration of the rfb_(sonnei) region in the SalI site. Arrowsdenote the direction of transcription of the indicated genes.

FIGS. 5A and 5B SDS-Page analysis of LPS minipreparations of O139 rfbclones in E. coli HB101 and V. cholerae CH19.

Panel A: silver stained. Panel B: Western blot using CH19-adsorbedpolyclonal rabbit O139-specific antiserum. Lanes: 1, Molecular weightmarkers; 2, CH19; 3, HB101 (pSSVI212-15) negative control; 4, MO45positive control; 5, HB101 (pSSVI212-3); 6, HB101 (pSSVI212-10); 7,HB101 (pSSVI212-13); 8, HB 101 (pSSVI212-16); 9, CH19 (pSSVI212-10); 10,CH19 (pSSVI212-13); 11, CH19 (pSSVI212-16)

FIG. 6: Restriction map of the integration vector pSSVI209.

Abbreviations and symbols are as in FIG. 3;

FIG. 7: Restriction map of the integration vector pSSVI199S.

Abbreviations and symbols are as in FIG. 3.

FIG. 8: Restriction map of the S. dysenteriae rfb/rfp loci integrationplasmid pSSVI208-2.

Abbreviations and symbols are as in FIG. 3. Box with: left stripes, rfplocus; right stripes, rfb locus.

FIG. 9: Restriction map of the E. coli rfe gene integration plasmidpSSV1219.

The arrows depict the direction of transcription of the indicated genes.White boxes: region homologous to CVD103-HgR genome; black box, rfegene; thin line+dotted boxes, pMAK700oriT vector. CmR, chloramphenicolresistance gene hlyB, 5′ end of the disrupted hlyB gene; hlyB′, 3′ endof the disrupted hlyB gene. Otherwise, as in FIG. 3.

FIGS. 10A-10C: SDS-PAGE analysis of O-PS expression in variousrfb_(Inaba) mutants of CVD103-HgR, CH3, and CH9, and in CH22.

Panels: A, silver stained gel; B, immunoblot with S. sonnei-specific MAbSh5S; C, immunoblot with the V. cholerae O-PS-specific MAb VCO4. Lanes:a, Molecular weight standard; b, CVD103-HgR; c, S. sonnei 482-79(pWR105); d, CH3; e, CH9; f, CH13; g, CH14; h, CH15; i, CH17; j, CH19;k, CH21; 1, CH22.

FIG. 11: Western blot analysis of LPS minipreparations of CH23, CH24,and V. cholerae carrier strains with plasmid-borne rfb/rfp loci alone ortogether with the plasmid-borne rfe gene.

Lanes: 1, molecular weight markers; 2, DH5α (pSS37); 3, CH19; 4, CH19(pSSVI208-2); 5, CH19 (pSSVI208-2/pRL100); 6, CVD-I⁻(pSSVI208-2/pRL100); 7, CH23; 8, CH23 (pSSVI219); 9, CH23 (pRL100), 10,CH24. Probing antibody: mouse S. dysenteriae O-PS-specific MAb MASD-1.

FIG. 12: Western blot analysis of LPS minipreparations of O139 rfbclones in E. coli HB101 and V. cholerae CH25.

Lanes: 1, Molecular weight markers; 2, HB101 (pSSVI215) negativecontrol; 3, MO45 positive control; 4 and 5, CH25; 6, HB101(pSSVI215-1₂); 7, HB101 (pSSVI215-2₃). Probing antibody: CH19-adsorbedpolyclonal rabbit O139-specific antiserum.

FIG. 13: Restriction map of plasmid pJMK10.

TABLE 1 Strains and plasmids Strains and plasmidsGenotype/Description^(a) Source Strains E.coli HB101 supE44 ara14 galK2lacY1 proA2 rpsL20 xyl-5 mtl-1 recA13 Δ(mcrC- Sambrook et al. Molecularcloning, 2nd edition, mrr) Cold Spring Harbor Laboratory Press, ColdSpring Harbor USA, (1989) DH5α F-Φ80dlacZΔM15 Δ(lacZYA-argF) U169 deorecA1 endA1 hsdR17 Sambrook et al. Molecular cloning, 2nd edition,(rK−,mK+) supE44 λ- thi1 gyrA96 relA1 phoA Cold Spring Harbor LaboratoryPress, Cold Spring Harbor USA, (1989) S17.1 thi-1 pro hsdR Tp^(r) Sm^(r)RP4-2[Tc::Mu(Km::Tn7)] Simon et al. Bio/Technology 1 (1983), :784S.sonnei 482-79 (pWR105) Phase I (smooth LPS) Sansonetti et al., Infect.Immun., 34 (1981), 75 V.cholerae CVD103-HgR O1 Classical Inaba. ΔctxAhlyA::mer (Hg^(R)) Ketley et al., FEMS Microbiol. Lett. 111 (1993), 15CVD111 O1 El Tor Ogawa. Δ(ctxA zot ace) hlyA::(ctxB mer) (Hg^(R)) M. M.Levine, pers. communication M045 wild type O139. Reference epidemicstrain Madras, India CH3 ΔctxA hlyA::mer hlyA::rfb/rfc_(sonnei) Viretand Favre, Biologicals 22 (1994), 361 CH9 ΔctxA hlyA::merhlyA::rfb/rfc_(sonnei)hlyB::rfa_(R1) Viret and Favre, Biologicals 22(1994), 361 CH13 CH3 ΔrfbDEGHI Present invention CH14 CH9 ΔrfbDEGHIPresent invention CH15 CVD103-HgR ΔrfbN Present invention CH17 CH9 ΔrfbNPresent invention CH19 CVD103-HgR ΔrfbAB Present invention CH21 CH9ΔrfbAB Present invention CH22 CH19 hlyA::rfb/rfc_(sonnei) Presentinvention CH23 CH19 hlyA::rfb_(dysenteriae) Present invention CH24 CH19hlyA::rfb_(dysenteriae)hlyA::rfe Present invention CH25 CH19hlyA::rfb_(O139) Present invention CH30 CVD111 ΔrfbAB Present inventionPlasmids pLAFR5 Broad host range cosmid vector 21.5 kb Keen et al., Gene70 (1988),.191 PMTL22p high-copy number general purpose plasmid vectorChambers et al., Gene 68 (1988), 139 pMAK700 low-copy numberthermosensitive suicide vector Hamilton et al., J. Bacteriol. 171(1989), 4617 pJFF350 transposon delivery vector with oriT sequenceFellay et al., Gene 76 (1989), 215 pGB2 low copy number general purposecloning vector Churchward et al., Gene 31 (1984), 165 pSSVI186-1 PlasmidpUC21 carrying the Sce-Km cassette Viret, BioTechniques, 14 (1993), 325pSS37 pACYC184 carrying the rfb and rfp loci of S.dysenteriae 1 Sturm etal. Microb. path. 1 (1986), 289 pRL100 plasmid bearing the rfe gene fromE. coli Meier-Dieter et al., J. Biol. Chem., 267 (1992), 746 pJMK10^(b)pUC19 carrying a 9.9 kb fragment with the hlyA-hlyB region fromV.cholerae 569B (wild type) interrupted by a 4.22 kb fragment bearingKetley et al., FEMS Microbiol. Lett. 111 (1993), 15 the mer operon(mercury resistance genes) pMAK700oriT Mobilizable suicide vector.pMAK700 with 0.75 kb oriT EcoRI-BamHI Present invention fragment frompJFF350 pSSVI255-3 rfb_(Inaba) locus cloned into pLAFR5 Presentinvention pSSVI255-5 rfb_(Inaba) locus cloned into pLAFR5 Presentinvention pSSVI255-7 rfb_(Inaba) locus cloned into pLAFR5 Presentinvention PSSVI205-1 pMAK700oriT carrying the entire insert ofpSSVI255-7 from which the Present invention three internal SacIfragments were deleted. The insert contains also, ca., 1 kb of pLAFR5DNA PSSVI205-2 same as pSSVI205-1 but insert in opposite orientationPresent invention pSSVI255-12 PMAK700oriT carrying the HindIII-SalIfragment of pSSVI255-7 at Present invention coordinates 8420-11730 fromwhich the central BamHI fragment was deleted pSSVI255-19 pMAK700oriTcarrying the ClaI fragment from pSSVI255-7 at Present inventioncoordinates 15770-21030 from which the central SalI fragment was deletedpSSVI255-20 pMAK700oriT carrying the SacI-BamHI fragment from pSSV1255-7at Present invention coordinates 5000-10340 from which the centralHindIII fragment was deleted pSSVI199S pMAK700oriT carrying thehlyA::mer locus, coordinates 0-5900 from Present invention plasmidpJMK10, added with a 2kb PCR fragment adjacent to the 5′-end of hlyA. Anextra SalI cloning site was created 345 bp downstream of merA pSSVI201-1pSSVI199S carrying the the rfb/rfc_(sonnei) locus Present inventionpSSVI212-13 pLAFR5 with the O139 rfb locus from V.cholerae O139 strainMO45 Present invention pSSVI209 pMAK700oriT carrying the hlyA″-hlyBfragment from pJMK10 Present invention (coordinates 5900-9900) completedwith the mer cassette (pJMK10 coordinates 1680-5900) in reverseorientation pSSVI220 pSSVI209 with the NotI fragment from pSSVI212-3cloned blunt into the Present invention Sall site pSS37-1K pGB2 with theXbaI-EcoRV 13.5 kb fragment from pSS37 carrying the Present inventionrfb/rfp loci of S.dysenteriae 1 cloned blunt together with the Sce-Kmcassette from plasmid pSSVI186-1 pSSVI208-1K pSSVI199S carrying therfb/rfp loci from S. dysenteriae together with the Present inventionSce-Km cassette from pSSVI186-1 pSSVI208-2K Same as pSSVI208-1K butrfb/rfp loci from S. dysenteriae and Sce-Km Present invention cassettein reverse orientation pSSVI208-2 pSSVI208-2K from which the Sce-Kmcassette was excised Present invention pMAK/hlyA pMAK700 oriT bearingthe hlyA″-hlyB fragment from pJMK10 Present invention (coordinates5900-9900) pSSVI219 pMAK/HlyA bearing the Xmal-Clal fragment from pRL100containing the Present invention rfe gene from E. coli ^(a)Coordinatesfor pSSVI255-7 are given in FIG. 1. ^(b)Coordinates for pJMK10 are givenin FIG. 13.

TABLE 2 Phenotypic characterization of CVD103-HgR and Inaba LPS mutantscellular phenotype affected single Strain function medium^(a)Motility^(b,e) cells^(e) filaments^(e) aggregates^(d,e) CVD103-HgR NoneCF +++ +++ − − LB +++ +++ − − BHI ++ +++ − − CH13 O−antigen CF − ++ ++ −transport, LB + +++ + + synthesis BHI − ++ + + CH14 O−antigen CF − +++++ − transport, LB + ++ +++ + synthesis BHI − + +++^(c) − CH15perosamine CF − ++ +++ +++ modification LB + + + +++ BHI + ++ +++ ++CH17 perosamine CF − + +++^(c) − modification LB ++ + +++ + BHI + +++++^(c) − CH19 perosamine CF − +++^(f) + +++ synthesis LB + +++ − +++BHI + +++ − +++ CH21 perosamine CF − ++ +++^(c) − synthesis LB + ++ +++− BHI − + +++^(c) − CH22 perosamine CF ++ ++ ++ − synthesis LB +++ +++ +− BHI + +++ ++ − ^(a)The strains were grown to stationary phase at 30°C. in the indicated medium ^(b)Microscopically determined. ^(c)Mostfilaments consisted of ≧10 cells ^(d)Large clusters of adherent cells^(e) − not present + present in 1 to 20% of population ++ present in 20to 60% of population +++ present in 60 to 100% of population

TABLE 3 Antibody response following immunization with V. choleraestrains CH22 or CVD103-HgR. Immunizing Geometric mean antibodytiters^(b) strain^(a) S. sonnei phase 1 LPS V. cholerae Inaba LPS NONE<10 <10 CH2 3′313 <14 (650-10′200) (<10-71)   CVD103-HgR <10 260 (57-730) ^(a)Groups of seven mice were immunized intramuscularly (IM)at days 0 and 14 with 5 × 10⁷ heat inactivated cell. A booster dose wasgiven intraperitoneally on day 21. Control mice were not immunized. Allmice were sacrificed on day 28. ^(b)Sera were tested individually forLPS-specific antibodies using purified S. sonnei phase 1 or V. choleraeInaba as coating antigens in an ELISA assay. Titers are expressed as thegeometric mean (range) of the reciprocals of the highest dilutionresulting in an OD_(405 nm) of 0.4.

What is claimed is:
 1. A live attenuated vaccine strain selected fromthe group consisting of Escherichia coli, Salmonella typhi, Vibrocholerae and Shigella, wherein said strain is unable to expresshomologous O-polysaccharide due to the introduction, by recombinanttechniques, of a 1.2 kb deletion that spans the junction of the rfbA andrfbB genes, and expresses at least one heterologous O-polysaccharidegene in such a way that said heterologous gene expresses a heterologousO-polysaccharide that is covalently linked to a lipopolysaccharide core.2. The live attenuated vaccine strain according to claim 1, wherein said1.2 kb deletion is a HinDIII deletion.
 3. The live attenuated strainaccording to claim 1, wherein said heterologous O-polysaccharide gene isintegrated into a chromosomal locus selected from the group consistingof hlyA, hlyB ctxA, rfbA, rfbB, and rfbA and rfb.
 4. The live attenuatedvaccine strain according to claim 1, wherein said strain is combinedwith a pharmaceutically acceptable carrier.
 5. The live attenuatedvaccine strain according to claim 1, wherein said strain is combinedwith a buffer for neutralizing gastric acidity.
 6. The vaccine strain ofclaim 1, wherein said heterologous O-polysaccharide gene is present on aplasmid vector or stably integrated into the chromosome of said strainat a defined integration site which is non-essential for inducing aprotective immune response by the carrier strain, said definedintegration site being a homologous genetic region corresponding to thegenetic region flanking said heterologous O-polysaccharide gene.
 7. Thevaccine strain of claim 6, wherein said heterologous O-polysaccharidegene is integrated into a chromosomal locus selected from the groupconsisting of hlyA, hlyB, ctxA, rfbA, rfbB, and rfbA and rfbB.
 8. Thevaccine strain of claim 6, wherein the strain is a S. typhi strain andwherein said heterologous O-polysaccharide gene is integrated into agene selected from the group consisting of the H₂S production gene, ilv,viaB, and htpR genes involved in virulence traits and genes involved inthe synthesis of aromatic acids.
 9. The vaccine strain according to anyone of claims 6 or 7, wherein rfa, rfe, and rfp genes are integrated intandem into a single chromosomal site or independently integrated intoindividual sites.
 10. The strain according to claim 9, wherein the rfagenes encode the Ra, R1, R2, R3, R4, K-12 or B LPS core.
 11. The strainaccording to claim 9, wherein the rfa genes encode the R1 core.
 12. Thelive attenuated vaccine strain which is Vibrio cholerae CH21, identifiedby the accession number DSM13421.
 13. The Vibrio cholerae vaccinecarrier strain, CH19, identified by the accession number DSM13420.
 14. Alive attenuated vaccine comprising the vaccine strain of claim 12,wherein said strain is combined with a pharmaceutically acceptablecarrier.
 15. A live attenuated vaccine comprising the vaccine strain ofclaim 12, wherein said strain is combined with a buffer for neutralizinggastric acidity.
 16. A live attenuated vaccine comprising the vaccinestrain of claim 12, wherein said vaccine is delivered in a viable stateto the intestinal tract.
 17. The live attenuated vaccine of claim 12 forimmunization of a mammalian subject in need thereof against agram-negative enteric pathogen selected from the group consisting ofShigella sonnei and Vibrio cholerae.
 18. The live attenuated vaccine ofclaim 12 for oral or intranasal administration.
 19. The live attenuatedvaccine strain according to claim 1, wherein said V. cholerae strain isselected from the group consisting of O1 V. cholerae and O139 V.cholerae.
 20. The live attenuated vaccine strain according to claim 1,wherein said V. cholerae strain is O139 V. cholerae selected from thegroup consisting of CVD112 and Bengal-15, or O1 V. cholerae selectedfrom the group consisting of CVD103, CVD103-HgR, CVD110, CVD111 andPeru-14.
 21. The vaccine strain of claim 1, wherein said heterologousO-polysaccharide gene is integrated into a chromosomal locus selectedfrom the group consisting of hlyA, hlyB and ctxA.
 22. A method forimmunizing against a gram-negative enteric pathogen selected from thegroup consisting of Shigella sonnei and Vibrio cholerae comprisingadministering the vaccine strain of claim
 12. 23. A method forimmunization against an enteric infection caused by a gram negativebacterial pathogen selected from the group consisting of Escherichiacoli, Salmonella typhi, Vibrio cholerae and Shigella comprisingadministering the live attenuated vaccine strain of claim 1, whereinsaid vaccine strain expresses the heterologous O-polysaccharide of thecorresponding said bacterial pathogen.